Download ELECTRON-MICROSCOPE ILLUSTRATIONS OF DIVISION IN

ELECTRON-MICROSCOPE ILLUSTRATIONS OF DIVISION
I N M Y C O B A C T E R I U M LEPRAE
ROSAP. EDWARDS
Department of Human Anatomy, University of Oxford
PLATESXLIX-LII
MYCOBACTERIUM
L E P R A E has never convincingly been grown in vitro, so
that its morphology must be studied in sections of infected tissues.
The organism grows extremely slowly in its human host and electron microscopy has
shown only a few instances of dividing bacteria and those only in a late stage of division
(Imaeda, Convit and Lapenta, 1963; Brieger and Allen, 1964; Klingmuller and Orfanos,
1966). Even in tissues from patients with severe untreated lepromatous leprosy, in which
there are very numerous bacteria, dividing organisms are seldom seen. This has led some
workers (Chatterjee, 1965) to suggest that the dividing form of the organism is a " soft
form ",or L-form, and that the stainable acid-fast organisms visible in tissues are some sort
of end-product, but do not themselves multiply. However, it has recently been demonstrated
that if mice whose immunological capacity has been reduced by thymectomy and total
body irradiation are given an inoculation of Myco. Zeprae, the bacteria multiply to give
very high yields (Gaugas, 1967; Rees et al., 1967). In many diagnosed human patients the
bacteria have almost reached the stationary phase of growth, but mouse tissues may be
sampled during the logarithmic phase, when the number of dividing micro-organisms is
correspondingly greater.
The morphology of Myco. Zeprae from tissues of such mice and the changes
found at various stages in the cycle of division have been observed in the
electron microscope. These findings are reported here, together with a few
observations of division in bacteria from patients with untreated lepromatous
leprosy.
MATERIALS
AND METHODS
Tissues were examined from three mice treated as detailed in the table and also from skin
and radial nerve biopsies from patients with untreated lepromatous leprosy. The human
material was fixed by immersion in either 4 per cent. neutral formaldehydesolution (Richardson, 1960) or in 3 per cent. glutaraldehyde in phosphate buffer (pH 7.2). The majority of
tissues were post-fixed in 1 per cent. phosphate-buffered osmium tetroxide (pH 7.2), embedded
in Araldite, sectioned, and stained in uranyl acetate followed by lead citrate. Foot-pad
tissues from one mouse, however, were divided into two. One part was processed by the
bacterial fixation technique of Kellenberger, Ryter and S6chaud (1958), and the other was
fixed in 2.5 per cent. glutaraldehydein 0 . 0 8 5 cacodylate
~
buffer (Glauert and Thornley, 1966),
washed in 5 per cent. buffered (pH 7-4) sucrose containing calcium chloride, and post-fixed
in osmium tetroxide buffered to pH 6.1 as recommended by Kellenberger et al. Both parts
were then treated in bulk with uranyl acetate, embedded in Araldite, sectioned and stained
with lead citrate. The material was examined either at 60 kV on a Siemens Elmiskop I or
at 80 kV on an Elmiskop 101.
Received 21 Oct. 1969; accepted 23 Jan. 1970.
J. MED. MICROBIOL.-VOL.
3 (1970)
493
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
494
ROSA P. E D WARDS
TABLE
Growth of Mycobacterium leprae in thymectomised and total-body
irradiated mice examined with the electron microscope
Source of
myco bacteria
Method of
inoculation
into mouse
Number of
bacteria
inoculated
Tissues
Survival
examined with
time of
electron
mouse (mth) microscope
Fixation of
tissues for
electron
microscopy
Passed from
man through
mouse
Intravenous
(tail vein)
3.1 x 108
12
Passed from
man through
mouse
Intravenous
(tail vein)
3x 109
9
Foot-pad
Glutaraldehyde
3 per cent.
Into hypodermis
in both hind
foot-pads
IX 105
10
Foot-pad
Glutaraldehyde
method of
Kellenberger
et al.
Neutral fomalPeripheral
dehyde 4 per
1ymph-nodes
cent.
Resting phase
RESULTS
Cell wall and surrounding zone. The cell wall is composed of two layers.
The inner layer (figs. l a and 2) is moderately electron-dense and sometimes
cannot be distinguished from the outer layer of the plasma membrane. The
outer layer is electron-transparent and is made visible in the specimens by a
tenuous electron-dense layer on its outer side. The zone between the bacterial
cell wall and the host cell cytoplasm presents a wide range of appearances.
The wall may be in direct contact with the host cytoplasm (figs. l a and 2) or
separated from it by a membrane (figs. l b and 3). It is more often surrounded
by a zone of low electron density, which envelops more than one bacterium
and often contains amorphous electron-dense grains or strands. This zone is
either in contact with the host-cell cytoplasm (figs. l c and 4) or the junction is
marked by a membrane (figs. Id and 5) (Imaeda et al., 1963; Imaeda, 1965).
Cell walls persist even when the cytoplasm has degenerated and the bacteria
are presumably dead (fig. 6).
Plasma membrane. This consists of two electron-dense layers separated
by an electron-transparent zone; each is approximately 3 nm in width. From
statistical analysis it is clear that there is no significant difference in the width
of the three layers of the plasma membrane. It is normally in contact with
both the cell wall and the cytoplasm, but perfect sections of whole bacilli
illustrating this are rare, for some separation, presumably due to a minimal
degree of plasmolysis, is usually present (fig. 7). In some cases extreme plasmolysis may follow the use of a hypertonic fixative (fig. 8).
Cytoplasmic contents. Mesosomes are not always visible and their position
is variable. They are invaginations of triple-layered membranes similar in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
ELECTRON MICROSCOPY OF MYCOBACTERIUM LEPRAE
495
appearance to the plasma membrane, with which, depending upon the plane
of section, they are sometimes found to be in continuity (fig. 9). I n some
bacteria membrane-bounded vesicles were seen lying in the space between the
plasma membrane and the cell wall (fig. 8). These may be mesosomes or parts
of them that have been extruded during plasmolysis (FitzJames, 1964; Weibull,
1965).
.
*
. .. .
I
FIG. 1.-Diagrams of four different appearances of the cell wall of Mycobacteriurn feprae and its
surrounding zone shown in figs. 2-5. (a) Appearance of the baci{lus Iying in direct contact
with the host-cell cytoplasm, as in fig. 2. (b) Appearance with a membrane ”, as in fig. 3.
(c) Appearance with a zone of low electron density surrounding the bacillus and making direct
contact with the host-cell cytoplasm, as in fig. 4. ( d ) Appearance with a zone,pf low electron
density surrounding the baciIlus and enclosed by a “junctional membrane , as in fig. 5.
1 = Bacterial cytoplasm. 2 = Plasma membrane, consisting of two dense layers on either
side of a less dense one. 3 = EIectron-dense layer of cell wall. 4 = Electron-transparent layer
of cell wall. 5 = Tenuous electron-dense zone outside cell wall. 6 = Host-cell cytoplasm.
7 = Membrane, possibly derived from the unit membrane of the cell wall when the bacillus
was phagocytosed. 8 = Zone of low electron density. 9 = Junctional membrane, formed
either at the interface of the host-cell cytoplasmic ground-substance and zone of low electron
density or as an extension of a lysosomal membrane.
The nucleoid appears as an electron-transparent zone containing numerous
fine electron-dense fibrils which may form aggregations (fig. 7) depending
upon fixation (Fuhs, 1965). The cytoplasin also contains granules of variable
electron density; some resemble polyphosphate granules seen in other mycobacteria, whilst others (fig. 10) appear to be lipid granules (Imaeda et al.,
1963).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
ROSA P. ED WARDS
496
Dividing bacteria
Division of bacteria in mice. In the earliest stage the plasma membrane
forms a slight concavity and two new electron-dense cell-wall layers extend
from it to the original cell wall. An electron-transparent layer lies between
(a)
FIG. 1 1 .-Diagrams of different stages of division of a Mycobacterium leprae bacillus as shown in
figs. 12-19. (a) The plasma membrane forms a concave ingrowth (figs. 12 and 18), and two
new electron-dense cell-wall layers separated by an electron-transparent layer are formed between
it and the original cell wall (fig. 12). (b) The annular concavity of the plasma membrane and
its contained cell walls grows centripetally until only a narrow cytoplasmic bridge remains
(fig..15). (c) Division is almost complete; the cytoplasmic bridge has been broken, but the
original electron-dense cell-wall layer still present joins the lateral cell walls of the daughter
bacilli (figs. 16 and 19). ( d ) The daughter bacilli have rounded ends; the electron-transparent
layer between is continuous with that around the cells (fig. 17). 1 = Bacterial cytoplasm.
2 = Plasma membrane consisting of two denser layers with a less dense layer between.
3 = Electron-dense layer of the cell wall. 4 = Electron-transparent layer of the cell wall.
5 = Tenuous electron-transparent layer outside the cell wall. 6 = Host-cell cytoplasm.
the dense layers (figs. l l a and 12). Later both plasma membrane and cell
walls are seen nearer the centre (" centripetal annular growth ") (figs. 1 l a and
13) forming perforated diaphragms through which there is a cytoplasmic
bridge (figs. 1 l a and 14). Still later the bridge is very constricted and, because
of the plane of the section and depth of field, the new electron-dense cell-wall
layers appear to form complete septa (figs. 1 l b and 15). Even when division
is complete and only an electron-transparent zone separates the newly formed
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
EDWARDS
ELECTRON
MICROSCOPY
OF
MY COB.4CTERIC .21
L E P RAE
Frc. 2.-Edge
of a Mycobacterium l c p m
bacillus (left) in a mouse lymph-node. See
fig. l a for identification of the structures.
Y 300.000.
FIG. 3.-Edge of a bacillus (left) in human skin.
See fig. Ib. x 370,000.
FIG.4.-Bacillus in a mouse lymph-node. See
fig. Ic. X 50,000.
FIG. 5.-Bacillus in a mouse lymph-node. See
fig. Id. Y 40,000.
All specimens fixed with formaldehyde. unless stated otherwise, and photographed
with the electron microscope.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
EDWARDS
PLATEL
ELECTRON
MICROSCOPY OF M ~ , C O B A CITCE
MRI_ L P R A L'
P-
c-
FIG. 6.-Cell wall and residual cytoplasm of a
bacillus in human skin. x 100,000.
NF
7.-Separation of the cell wall from
the plasma nienibrane (plasniolysis) of a
bacillus in a mouse lymph-node. N F
Nucleoid fibrils; A
fibril aggregates;
C = cell wall; P = plasma membrane.
64,000.
FIG.8.-Menibrane-bounded
vesicles in the
space between the plasma membrane and
the cell wall (plasniolysis) cf a bacillus in a
human nerve. Glutaraldehyde fixation.
x 144,000.
FrG.
FIG.9.-Mesosome
in continuity with the
plasma membrane in a bacillus in a nioiise
foot-pad. G lutaraldehyde/Kellenberger fixation. x 160,000.
FIG. 10.-Granules in the cytoplasm of a
bacillus in a mouse lymph-node. M =
Mitochondrion; L = polyphosphate granule; P = lipid granule.
64,000.
7
~
t~
All specimens fixed with formaldehyde, unless stated otherwise, and photographed
with the electron microscope.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
PLATELI
EDWARDS
ELECTRON
MICROSCOPY
OF
M Y C O B A C T E R I LUEM
PRAE
FIG.12.-Initial stage of division of a bacillus
in a mouse lymph-node. ?C 85,000.
FIG.13.-Later stage of division of a bacillus
in a mouse lyniph-node, showing centripetal
annular growth. Y 65,000.
FIG. 14. - Cytoplasmic
bridge
between
daughter cells in a dividing bacillus in a
mouse lymph-node. x 180,000.
FIG. 15.-Narrower cytoplasmic bridge in a
dividing bacillus in a niouse lymph-node.
,< 85,000.
All specimens fixed with formaldehyde, unless stated otherwise, and photographed
with the electron microscope.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
PLATE
LII
EDWARDS
ELECTRON
MICROSCOPY OF MYCOBACTERIUM
LEPRAE
16.-Electron-transparent layer separating the daughter cells of a newly divided
bacillus in a mouse lymph-node. Note
the continuity of the peripheral cell wall.
x 230,000.
FIG.
FIG. I8.--Early
stage of division of a
bacillus in a human nerve. A niesosoiiie
lies in contact with the indented plasma
membrane. The overlapping images of
the plasma membrane are probably due
to the surface of both sides of the section
being stained. Glutaraldehyde fixation.
x 240,000.
FIG. 17.-Electron-transparent
layer surrounding the daughter cell of a newly
divided bacillus. Y 235,000.
FIG. 19.-Later
stage of division of a
bacillus in a human nerve. Mesosonial
vesicles are extruded in one daughter cell
(plasmolysis) (cf. fig. 16). Glutaraldehyde
fixation. Y 225.000.
All specimens fixed with formaldehyde, unless stated otherwise, and photographed
with the electron microscope
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
ELECTRON MICROSCOPY O F MYCOBACTERIUM LEPRAE
497
cells, the original electron-dense cell-wall layer is present (figs. 11c and 16).
Later this wall disappears and the daughter cells have rounded ends and the
electron-transparent layer between them is continuous with the electrontransparent layer of the cells themselves (figs 1Id and 17). These last two stages
in division are those most commonly found. Complete separation occurs later
still. In all the dividing bacteria observed, an electron-transparent region
separated the bacterial cytoplasm and plasma membrane from the newly formed
cell wall. This is thought to be an artefact caused by plasmolysis.
Division of bacteria in man. Very few dividing bacilli were seen, but the
morphological changes found were comparable with those seen in material
from mice. For example, in an early stage a mesosome was seen in contact
with the indented plasma membrane (fig. 18). In a later stage bacterial division
was nearly complete, though the original cell wall was still intact (figs. 1lc and
19). In one of the daughter cells the plasma membrane was almost complete,
but in the other, owing to plasmolysis, membrane-bounded mesosomal vesicles
had been extruded (fig. 19). Daughter cells, united only by the electrontransparent zone of their walls, were seen in a number of sections. A series of
drawings illustrating the process of division in Myco. Zeprae is shown in fig. 11.
DISCUSSION
Glutaraldehyde fixation followed by post-fixation either with osmium
tetroxide buffered at pH 7.2 or at pH 6.1 according to Kellenberger et aZ. (1958)
gave satisfactory pictures of both bacteria and the cells in which they were
situated. The use of Kellenberger’s method alone proved unsatisfactory for
the study of Mycobacterium Zeprae in tissues from mice. The use of formaldehyde
in 0 . 7 5 ~sucrose gave rise to plasmolysis and this enabled the plasma membrane to be more clearly observed. It also revealed some of the detailed structure of the vesicles in the space between the plasma membrane and the cell wall,
which may be expelled mesosomes (FitzJames, 1964; Weibull, 1965).
The ultrastructure of the cell wall of Myco. Zeprae was found to be similar
to that of other mycobacteria (Imaeda, Kanetsuna and Galindo, 1968), and
observations on the plasma membrane of Myco. Zeprae do not differ from those
of other workers who have studied mycobacteria (Imaeda and Ogura, 1963).
Comparable variations in the zone surrounding them have also been noted
previously (Imaeda, 1965).
During division of Myco. Zeprae it was observed that both components of
the cell wall grow inwards to form a septum. This process is the same as that
seen in other dividing mycobacteria (Imaeda and Ogura) and in other Grampositive bacteria (Ellar, Lundgren and Slepecky, 1967; Kakefuda, Holden
and Utech, 1967). In human material, bacteria that had reached the stage of
completion of the new cell wall, but had not separated, were frequently seen,
but other stages were rare. This implies that although bacteria may divide as
seldom as once in 2-3 wk, the actual process of formation of the cell wall
probably occurs relatively rapidly.
The intracytoplasmic membrane systems now generally referred to as
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
498
ROSA P . ED WARDS
mesosomes (FitzJames, 1960) are considered to be formed as invaginations of
the plasma membrane. In mycobacteria they are often very elaborate. Their
function is still uncertain; it seems likely, however, that they are concerned
among other things with the synthesis of the cell wall and nuclear segregation
during division (Ellar et aZ.; Ryter, 1967). Imaeda and Ogura suggested that
in Myco. Zepruernurium a precursor of the cell wall lies within the invagination
and is in contact with the cell wall through a narrow opening. The observations
recorded in the present paper are not at variance with the results of other
authors (Imaeda and Convit, 1962; Kakefuda et al.), but they do not contribute
to the question of whether or not mycobacteria contain more than one mesosome, or whether the mesosomes are invariably associated with cell division, or
have some additional function such as electron transport (van Iterson, 1965). It
should be noted, however, that in some instances mesosomes were found
apparently in close proximity to the site of initiation of division (fig. 18).
SUMMARY
The sequential changes occurring during bacillary division of Mycobacterium
Zeprae in infected foot-pads of mice and human skin and nerves were studied
with the electron microscope and found to be as follows : (1) a slight concavity
develops in the plasma membrane, (2) two new electron-dense cell-wall layers
are found between the concavity and the original cell wall, and an electrontransparent cell-wall layer appears between them, and gradually separates
them, and (3) the annular ingrowth of the plasma membrane and cell wall
proceeds until division is complete. There is no essential difference between
the behaviour of Myco. Zeprae and that of other mycobacteria, nor between
the behaviour in bacteria obtained from human biopsy specimens and bacteria
from infected mice. Although Myco. Zeprae multiplies slowly the actual process
'
of cell division may be relatively rapid.
I wish to thank Dr R. J. W. Rees, National Institute for Medical Research, Mill Hill,
Dr G. Weddell, Department of Human Anatomy, Oxford, and their colleagues for help
and encouragement, and Mr Derek Wood for technical assistance.
REFERENCES
BRIEGER,
E. M., AND ALLEN,
JENNIFER 1964. In Leprosy in theory and practice. ed. by
R. G . Cochrane and T. F. Davey,
Bristol, pp. 36-49.
CHATTERJEE,
B. R.
.
,
1965. Znt. J. Lepr., 33, Suppl., p. 551.
ELLAR,D. J., LUNDGREN,
D. G., AND 1967. J. Bact., 94, 1189.
SLEPECKY,
R. S.
FITZJAMES,
P. C .
. 1960. J. Biophys. Biochem. Cytol., 8, 507.
. 1964. J. Bact., 87, 1483.
FUHS,G. W.
.
. 1965. Bact. Rev., 29, 277.
GAUGAS,
J. M. .
,
1967. Br. J. Exp. Path., 48, 417.
GLAUERT,
AUDREYM., AND THORNLEY,1966. J . Roy. Microsc. SOC.,85, 449.
MARGARET
J.
T.
. 1965. Znt. J. Lepr., 33, 669.
IMAEDA.
IMAEDA,T., AND CONVIT,
J.
. 1962. J . Bact., 83, 43.
9,
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
!ELECTRON MZCROSCOPY OF M YCOBACTERIUM LEPRAE
IMAEDA,
T., CONVIT,
J., AND LAPENTA,
P. 1963.
IMAEDA,
T., KANETSUNA,
F., AND GALINDO,1968.
B.
IMAEDA,
T., AND OGURA,
M..
1963.
KAKEFUDA,
T., HOLDEN,
J. T., AND UTECH, 1967.
N. M.
KELLENBERGER,
E., RYTER, A., AND 1958.
S~CHAUD,
J.
KLING~LLE
G.,
R ,AND ORFANOS,
C. . 1966.
REES, R. J. W., WATERS,M. F. R., 1967.
WEKKEL,A. G. M., AND PALMER,
ELISABETH
RICHARDSON,
K. C.
.
1960.
RYTER,A..
1967.
VAN ITERSON,
W.
1965.
WEIBULL,
C.
.
1965.
Int. J. Lepr., 31, 389.
J. Ultrustruct. Res., 25, 46.
J. Bact., 85, 150.
Zbid., 93, 472.
J. Biophys. Biochem. Cytol., 4, 671.
Arch. exp. Derm., 224, 373.
Nature, Lond., 215, 599.
J . Anat., 94, 457.
Folia microbiol., 12,283.
Bact. Rev., 29, 299.
J . Bact., 89, 1151.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 18:47:08
499